Due to rise in cardiovascular disease throughout the world, there is increasing demand for small diameter blood vessels as replacement grafts. In principle, the need for large number of autologous cells to populate those tissues can be met by adult mesenchymal stem cells (MSC), which show significant proliferation and differentiation potential. However, reports from several laboratories including ours showed that MSC originating from older donors suffer from limited proliferative capacity and significantly reduced differentiation potential. This is a major concern, as the patients mostly in need for vascular grafts are elderly. The current proposal seeks to address this challenge using a highly innovative approach that is based on preliminary data from our laboratory showing that the effects of aging can be reversed by expression of a single transcription factor, namely Nanog. Based on these results we propose the following specific aims. In the new aim 1 we will examine whether transient expression of Nanog is sufficient to reverse the effects of donor aging on proliferation and myogenic differentiation of MSC. In aim 2 we will investigate a novel hypothesis to uncover the mechanism through which Nanog enhances transcription of myogenic genes and contractile function. We will also employ oligonucleotide microarrays as well as high throughput epigenetic analysis to determine how Nanog restructures the chromatin and unravel novel genes that are differentially expressed due to Nanog expression. In aim 3 we will develop a novel strategy to deliver the Nanog protein into MSC in order to restore their lost function. Novel hypotheses will be addressed to engineer Nanog protein variants with enhanced transcriptional activity in order to maximize their potency in reversing the effects of senescence on MSC myogenic differentiation potential. Finally, in aim 4 we will examine the potential of the Nanog-treated stem cells for engineering small-diameter, functional blood vessels that could be implanted into the arterial system of a pre-clinical ovine animal model that was developed in our laboratory. Successful completion of this project will significantly enhance the therapeutic clinical potential of MSC from adult donors without genetic modification or reprogramming to the pluripotent state.